PROJECT SUMMARY Candida albicans is an important opportunistic pathogen for people living with HIV/AIDS and primarily causes mucosal diseases such as oropharyngeal candidiasis (OPC) and esophageal candidiasis (EC). In the era of antiretroviral therapy (ART), C. albicans remains the most common fungal pathogen affecting those with HIV/AIDS and OPC is one of the most common infections in HIV patients with mild-to-moderate immunosuppression (CD4 counts 200-500). The pathogenesis of OPC can be separated into two stages: 1) C. albicans adhesion/biofilm formation on the mucosal surface and 2) hyphae-mediated invasion of the epithelium and submucosal stroma with concomitant inflammation and tissue damage. Thus, OPC pathogenesis is dependent on three of the most important mediators of C. albicans virulence: adhesion, biofilm formation, and filamentation. Although each of these factors has been studied using specific C. albicans mutants, no systematic large-scale genetic analysis of OPC pathogenesis has been undertaken. The goal of this application is to define and characterize the transcriptional networks that underlay the ability of C. albicans to cause oral disease. Recently, we have pioneered the use of complex haploinsufficiency (CHI)-based genetic interaction analysis to understand the function of complex genetic networks. Here, we propose to use CHI analysis to test the hypothesis that Cbk1 represents a master regulator of transcriptional processes critical for OPC pathogenesis (Aim 1). In addition to this intra-pathway analysis, we will perform the first large-scale genetic screens for, and CHI analyses of, TF networks required for murine oral candidiasis (Aim 2) and in vivo filamentation (Aim 3). The screen in Aim 3 will utilize our novel intra-vital imaging assay to quantitatively differentiate between yeast and filamentous cells in the sub-epithelial stroma of mice. This screen will not only be the first in vivo C. albicans filamentation screen but also will allow us to distinguish TFs required for the invasion stage of OPC from TFs required for the adhesion/biofilm formation stage.